Orthogonal deblocking of nucleotides

ABSTRACT

A method including steps of (a) providing an array of sites, wherein each site comprises a mixture of different nucleic acid templates; (b) extending primers hybridized to the different nucleic acid templates at each of the sites with different nucleotide analogs having different reversible blocking moieties, respectively, thereby producing different primer extension products at each site; (c) detecting the different primer extension products to distinguish the different nucleotide analogs at each site; and (d) removing the different reversible blocking moieties from the primer extension products at each of the sites using a first treatment that is selective for a first of the different reversible blocking moieties and a second treatment that is selective for a second of the different reversible blocking moieties.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/739,587, filed Dec. 22, 2017, which application is a U.S. NationalStage Application filed under 35 U.S.C. § 371 claiming priority toInternational Application No. PCT/US2016/041568, filed Jul. 8, 2016,which application claims priority to U.S. Provisional Application Ser.No. 62/198,947, filed Jul. 30, 2015, the disclosures of which areincorporated herein by reference in their entirety.

BACKGROUND

This disclosure relates generally to nucleic acid analysis, and morespecifically to nucleic acid sequencing.

Currently available commercial platforms for sequencing DNA arerelatively costly. These platforms use a ‘sequencing-by-synthesis’approach, so called because DNA polymers are synthesized while detectingthe addition of each monomer (i.e. nucleotide) to the growing polymerstructure. Because a template DNA strand strictly directs synthesis of anew DNA polymer, one can infer the sequence of the template DNA from theseries of nucleotide monomers that were added to the growing strandduring the synthesis. The ability to detect monomer additions isfacilitated by specially engineered variants of the biochemicalcomponents that normally carry out DNA synthesis in biological systems.These engineered components are relatively expensive to make and areconsumed in relatively large amounts during sequencing-by-synthesis.Furthermore, monitoring the reaction uses relatively expensive hardwaresuch as lasers, detection optics and complex fluid delivery systems. Themost successful commercial platforms to date also require expensivereagents and hardware to amplify the DNA templates beforesequencing-by-synthesis can even begin. The complexity and expense ofthese platforms has hindered their use in some clinical and researchcontexts where there is a clear need for the technology.

Thus, there exists a need for improvements to sequencing-by-synthesisplatforms to make them more cost effective, rapid and convenient. Thepresent disclosure addresses this need and provides other advantages aswell.

BRIEF SUMMARY

The present disclosure provides a method for identifying nucleic acidtemplates. The method can include steps of (a) providing an array ofsites, wherein each site comprises a mixture of at least two differentnucleic acid templates; (b) extending primers hybridized to thedifferent nucleic acid templates at each of the sites with differentnucleotide analogs having different reversible blocking moieties,respectively, thereby producing different primer extension products ateach site; (c) detecting the different primer extension products todistinguish the different nucleotide analogs at each site; and (d)removing the different reversible blocking moieties from the primerextension products at each of the sites using a first treatment that isselective for a first of the different reversible blocking moieties anda second treatment that is selective for a second of the differentreversible blocking moieties. Optionally, the method can further include(e) repeating (b) through (d) to determine the sequence of differentnucleotide analogs added to each of the different extension products ateach of the sites.

Also provided is a method for sequencing nucleic acid templates that caninclude the steps of (a) providing an array of sites, wherein each siteincludes a first nucleic acid template and a second nucleic acidtemplate, wherein the first nucleic acid template has a sequence that isdifferent from the sequence of the second nucleic acid template, whereina first primer is bound to the first nucleic acid template, and whereina second primer is bound to the second nucleic acid template, areversible blocking moiety being attached to the second primer; (b)extending the first primer by addition of a first nucleotide analog thatis attached to a reversible blocking moiety, wherein the reversibleblocking moiety that is attached to the first nucleotide is differentfrom the reversible blocking moiety that is attached to the secondprimer; (c) selectively removing the reversible blocking moiety that isattached to the second primer while retaining the reversible blockingmoiety that is attached to the nucleotide analog that is added to thefirst primer; (d) extending the second primer by addition of a secondnucleotide analog that is attached to a reversible blocking moiety,wherein the reversible blocking moiety that is attached to the firstnucleotide analog is different from the reversible blocking moiety thatis attached to the second nucleotide analog; and (e) detecting thenucleotide analog that is added to the first primer and the nucleotideanalog that is added to the second primer, at each of the sites, therebydetermining the different sequences of the first template and the secondtemplate at each of the sites. Optionally, the method can furtherinclude steps of (f) selectively removing the reversible blocking moietythat is attached to the first nucleotide analog that is added to thefirst primer while retaining the reversible blocking moiety that isattached to the second nucleotide analog that is added to the secondprimer; (g) extending the first primer, after (f), by addition of athird nucleotide analog that is attached to a reversible blockingmoiety; (h) selectively removing the reversible blocking moiety that isattached to the second nucleotide analog that is added to the secondprimer while retaining the reversible blocking moiety that is attachedto the first nucleotide analog that is added to the first primer; (i)extending the second primer, after (h), by addition of a fourthnucleotide analog that is attached to a reversible blocking moiety,wherein the reversible blocking moiety that is attached to the thirdnucleotide analog is different from the reversible blocking moiety thatis attached to the fourth nucleotide analog; and (h) detecting thenucleotide analog that is added to the first primer in (g) and thenucleotide analog that is added to the second prime in (i), at each ofthe sites, thereby determining the different sequences of the firsttemplate and the second template at each of the sites. Optionally, steps(f) through (h) can be repeated.

Also provided is a method for sequencing nucleic acid templates that caninclude the steps of (a) providing an array of sites, wherein each siteincludes a first nucleic acid template and a second nucleic acidtemplate, wherein the first nucleic acid template has a sequence that isdifferent from the sequence of the second nucleic acid template, whereina first primer is bound to the first nucleic acid template, a reversibleblocking moiety being attached to the first primer, wherein a secondprimer is bound to the second nucleic acid template, a reversibleblocking moiety being attached to the second primer, and wherein thereversible blocking moiety that is attached to the first primer isdifferent from the reversible blocking moiety that is attached to thesecond primer; (b) selectively removing the reversible blocking moietythat is attached to the first primer while retaining the reversibleblocking moiety that is attached to the second primer; (c) extending thefirst primer by addition of a first nucleotide analog that is attachedto a reversible blocking moiety; (d) selectively removing the reversibleblocking moiety that is attached to the second primer while retainingthe reversible blocking moiety that is attached to the nucleotide analogthat is added to the first primer; (e) extending the second primer byaddition of a second nucleotide analog that is attached to a reversibleblocking moiety, wherein the reversible blocking moiety that is attachedto the first nucleotide analog is different from the reversible blockingmoiety that is attached to the second nucleotide analog; and (f)detecting the nucleotide analog that is added to the first primer andthe nucleotide analog that is added to the second primer, at each of thesites, thereby determining the different sequences of the first templateand the second template at each of the sites.

The present disclosure further provides a nucleic acid array thatincludes a plurality of sites on a solid support, wherein each siteincludes a first nucleic acid template and a second nucleic acidtemplate, wherein the first nucleic acid template has a sequence that isdifferent from the sequence of the second nucleic acid template, whereina first primer is bound to the first nucleic acid template, a firstreversible blocking moiety being attached to the first primer, wherein asecond primer is bound to the second nucleic acid template, a secondreversible blocking moiety being attached to the second primer, andwherein the first reversible blocking moiety is different from thesecond reversible blocking moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cycle diagram for sequencing by synthesis carried outsimultaneously for a mixture of two templates at a site of an array,wherein subsets of label moieties and deblocking moieties that arepresent on the same extension products are cleaved simultaneously.

FIG. 2 shows a cycle diagram for sequencing by synthesis carried outsimultaneously for a mixture of two templates at a site of an array,wherein label moieties that are present on different extension products(i.e. produced in both R1 and R2 reactions) are cleaved simultaneously.

DETAILED DESCRIPTION

This disclosure provides a method for high density detection of nucleicacids. Particular embodiments of the methods of the present disclosureexploit known techniques for manipulating and detecting nucleic acids.However, improvements set forth below provide orthogonal processing suchthat the density of information obtained from use of these techniques isincreased.

The example of a primer extension-based detection technique isillustrative of the increased density of information that can beobtained. Specifically, a target sequence of a nucleic acid can behybridized to a primer and the primer extended by a DNA polymerase toadd a labeled nucleotide analog. An array format can be used withmultiple sites, each site having a single target sequence that differsfrom the target sequences present at other sites. Optionally severaldifferent nucleotide analog species, each having a distinguishablelabel, are used as well. Primer extension results in recruitment of thelabeled nucleotide analog to the nucleic acid having the targetsequence. In an array format, where different labeled nucleotide analogsare used, one can distinguish the label that is recruited to each site,and use this information to identify the target nucleic acid at thatsite. The density of information obtained from this array format is onetarget sequence identified per site.

In an orthogonal format of the present disclosure, each site of thearray can contain a mixture of two or more different target sequencesthat are simultaneously treated (e.g. with chemical reagents or physicalmanipulations) and simultaneously observed (e.g. with a detector havingresolution that is too low to spatially resolve nucleic acids in themixture). Nonetheless, the orthogonal treatments set forth hereinproduce differential effects that allow the different target sequencesto be distinguishable from each other. In this case the informationderived from the array can be at least doubled. Two different primerscan be delivered to an array and due to differential complementary theywill hybridize to the two different template sequences, respectively, ateach individual site. The first primer can have a first reversibleblocking moiety that prevents it from being extended until a firstdeblocking treatment is applied, and the second primer can have a secondreversible blocking moiety that prevents it from being extended until asecond deblocking treatment is applied. In this example, the firstdeblocking treatment is selective for the first reversible blockingmoiety compared to a second reversible blocking moiety and the seconddeblocking treatment is selective for the second reversible blockingmoiety compared to the first reversible blocking moiety. This selectivedeblocking capability provides orthogonality such that the primers,although being exposed to each deblocking treatment, can be individuallymodified for extension and detection. Thus the two primers, and moresignificantly the templates that direct their extension, can bedistinguished by this chemical switching even though the moleculesthemselves may not be separated sufficiently to allow spatialdistinction using detection system in use.

The concepts of orthogonality exemplified above for a primerextension-based detection technique can be readily applied to asequencing-by-synthesis (SBS) technique. An exemplary cycle diagram fororthogonal SBS for two templates (R1 and R2) at a site is shown inFIG. 1. The first column in the diagram represents a treatment to whichthe array is exposed (i.e. both of the templates are exposed to thetreatment). The second and third columns indicate the effect of thetreatment on the first template and second template, respectively. Inthe first step of the first cycle, a mixture of primers is contactedwith the array which results in hybridization to respective primerbinding sites. Following step 1, the R1 primer that is hybridized to theR1 template is blocked by block1 and the R2 primer lacks a blockinggroup (optionally primer R2 can be blocked by an orthogonal blockingmoiety, block2). As such, the two primers can be separately extended.For example, in the second step of the first cycle, a nucleotide havingblock2 and label2 is delivered to the array, which results in selectiveextension of the unblocked R2 primer (i.e. the R1 primer is notextended). Then in the third step of the first cycle, the array can beexposed to a treatment that selectively deblocks the R1 primer, forexample, by cleavage of block1 (i.e. block2 is not cleaved). In thefourth step of the first cycle, a nucleotide having block1 and label1 isdelivered to the array, which results in selective extension of theunblocked R1 primer (i.e. the R2 primer is not extended). The array canthen be observed using a detection device such that the two labels canbe distinguished at each site. As exemplified in FIG. 1, cycles ofdelivering nucleotides that are blocked and labeled, cleaving blockinggroups and labels and detection can be repeated cyclically.

As exemplified above and in FIG. 1, the nucleotide analogs that are usedto extend the primers can include reversible blocking moieties that areselectively cleavable to provide orthogonal control through multiplecycles of a sequencing by synthesis process. Thus, the nucleotideanalogs can be provided in two sets: a first set having a firstreversible blocking moiety (e.g. the same as the reversible blockingmoiety on the first primer, block1) and a second set having a secondreversible blocking moiety (e.g. block2). In some embodiments, thenucleotide analogs in the first set can have labels that aredistinguishable from the labels on the nucleotide analogs in the secondset (e.g. a first set is indicated at label1 and a second set isindicated as label2 in FIG. 1). The resulting orthogonality inbiochemical reactivity and label management allows the two primerextension events to be distinguished from each other at each site of anarray. Thus, the two target sequences can be distinguishably detected.

It will be understood that other methods can also benefit fromorthogonal manipulation and detection as set forth in further detailbelow. Thus, the compositions, apparatus and methods set forth hereinneed not be limited to sequencing applications.

Orthogonality can be exploited to increase the density of informationacquisition by 2-fold or more. For example, greater than 2-fold increasein information density can be obtained by using greater than twoorthogonal reagent sets. As an example, 3 reagent sets can be usedincluding 3 different deblocking treatments that are each selective forthe primers and/or nucleotide analogs in one of the reagent sets.

As demonstrated above and as will be set forth in further detail below,the present disclosure provides the advantage of super-resolutionimaging of an array, whereby the number of simultaneously resolvabletarget sequences at a given site is greater than one. Super-resolutionimaging can provide the benefit of simultaneously distinguishing anumber of different target nucleic acids that is larger than the numberof sites on the array. Similarly, super-resolution is provided in thattwo different target sequences can be distinguished on a solid phasesubstrate using a detector that has a resolution that is lower than thespatial resolution that would otherwise be required to distinguish thetwo target sequences on the substrate.

In particular embodiments, this disclosure provides reagent and hardwareconfigurations for efficient nucleic acid detection. An exemplaryconfiguration uses fewer labels than the number of nucleotide analogspecies that is to be distinguished in a primer extension step. Forexample, four species of nucleotide analog can be distinguished based ondetection of a single label species. As set forth in further detailbelow, this can be achieved by using a first set of nucleotide analogsincluding the following four species: (1) a species having a firstlabel, (2) a species having a ligand, (3) a species having a cleavablelinkage to the first label, and (4) a species lacking any label orligand used in a subsequent step, wherein all four species have ablocking moiety that is selectively deblocked by a first treatment. Anorthogonal set of nucleotide analogs can include the following fourspecies (5) a species having a second label, (6) a species having amixture of the first and second labels, (7) a species having a cleavablelinkage to the second label, and (8) a species lacking any label orligand used in a subsequent step wherein all four species have ablocking moiety that is selectively deblocked by a second treatment.Specifically, the first treatment does not cause substantial deblockingof the first set of nucleotide analogs and the second treatment does notcause substantial deblocking of the orthogonal set of nucleotideanalogs.

The species within each set above can be distinguished from each otherbased on a proper accounting of what labels appear or disappear afterspecific fluidic steps and the two orthogonal sets of nucleotide analogscan be distinguished based on the two different labels. Morespecifically, species (1) and (5) can be distinguished from each otherbased on different labels and from all other species due to theirappearance after an initial labeling step and their resistance torespective cleaving agent; species (2) can be distinguished based onappearance of label after incubation with a labeled receptor; species(3) and (7) can be distinguished from each other based on the differentlabels and are distinguished from all other species based upon initialappearance of the label and then disappearance after treatment with arespective cleavage reagent; species (6) can be distinguished from allother species based on the presence of both labels at an intensity thatis half the intensity for fully labeled species; and species (4) and (8)can be distinguished based on inference from a lack of any other speciesin the respective sets having been detected. Many other configurationsare possible to alter the number of labels, number of fluidicmanipulations during a detection phase and/or the complexity of thedetection device to distinguish a certain number of labels. As such, theconfiguration can be tailored to suit a particular approach orapplication.

Terms used herein will be understood to take on their ordinary meaningunless specified otherwise. Examples of several terms used herein andtheir definitions are set forth below.

As used herein, the term “amplicon,” when used in reference to a nucleicacid, means the product of copying the nucleic acid, wherein the producthas a nucleotide sequence that is the same as or complementary to atleast a portion of the nucleotide sequence of the nucleic acid. Anamplicon can be produced by any of a variety of amplification methodsthat use the nucleic acid, or an amplicon thereof, as a templateincluding, for example, polymerase extension, polymerase chain reaction(PCR), rolling circle amplification (RCA), ligation extension, orligation chain reaction. An amplicon can be a nucleic acid moleculehaving a single copy of a particular nucleotide sequence (e.g. a PCRproduct) or multiple copies of the nucleotide sequence (e.g. aconcatameric product of RCA). A first amplicon of a target nucleic acidis typically a complementary copy. Subsequent amplicons are copies thatare created, after generation of the first amplicon, from the targetnucleic acid or from another amplicon. A subsequent amplicon can have asequence that is substantially complementary to the target nucleic acidor substantially identical to the target nucleic acid.

As used herein, the term “array” refers to a population of sites thatcan be differentiated from each other according to relative location.Different molecules that are at different sites of an array can bedifferentiated from each other according to the locations of the sitesin the array. An individual site of an array can include one or moremolecules of a particular type. For example, a site can include a singletarget nucleic acid molecule having a particular sequence or a site caninclude several nucleic acid molecules having the same sequence (and/orcomplementary sequence, thereof). Alternatively a site can include amixture of target nucleic acid sequences, for example, such thatindividual molecules contain two or more different molecules each orsuch that two or more molecules each contain a single target sequence ofthe mixture. The sites of an array can be different features located onthe same substrate. Exemplary features include without limitation, wellsin a substrate, beads (or other particles) in or on a substrate,projections from a substrate, ridges on a substrate or channels in asubstrate. The sites of an array can be separate substrates each bearinga different molecule. Different molecules attached to separatesubstrates can be identified according to the locations of thesubstrates on a surface to which the substrates are associated oraccording to the locations of the substrates in a liquid or gel.Exemplary arrays in which separate substrates are located on a surfaceinclude, without limitation, those having beads in wells.

As used herein, the term “attached” refers to the state of two thingsbeing joined, fastened, adhered, connected or bound to each other. Forexample, an analyte, such as a nucleic acid, can be attached to amaterial, such as a gel or solid support, by a covalent or non-covalentbond. A covalent bond is characterized by the sharing of pairs ofelectrons between atoms. A non-covalent bond is a chemical bond thatdoes not involve the sharing of pairs of electrons and can include, forexample, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilicinteractions and hydrophobic interactions. A nucleic acid can beattached to a solid support via a gel coating on the solid support.

As used herein, the term “blocking moiety,” when used in reference to anucleotide analog, means a part of the nucleotide analog that inhibitsor prevents the nucleotide analog from forming a covalent linkage to asecond nucleotide analog. For example, in the case of nucleotide analogshaving a pentose moiety, a blocking moiety can prevent formation of aphosphodiester bond between the 3′ oxygen of the nucleotide analog andthe 5′ phosphate of the second nucleotide analog. The blocking moietycan be part of a nucleotide analog that is a monomer unit present in anucleic acid polymer or the blocking moiety can be a part of a freenucleotide analog (e.g. a nucleotide triphosphate). The blocking moietythat is part of a nucleotide analog can be reversible, such that theblocking moiety can be removed or modified to render the nucleotideanalog capable of forming a covalent linkage to a second nucleotideanalog. Particularly useful reversible blocking moieties are phosphates,phosphoesters, alkyl azides, acetals, esters, ethers or the like.Further examples of reversible blocking moieties that can be used areset forth below and in references incorporated by reference herein asset forth below. In particular embodiments, a blocking moiety, such as areversible blocking moiety, can be attached to the 3′ position or 2′position of a pentose moiety of a nucleotide analog.

As used herein, the term “cluster,” when used in reference to nucleicacids, refers to a population of the nucleic acids that is attached to asolid support to form a feature or site. The nucleic acids are generallyof a single species, thereby forming a homogenous cluster. However, insome embodiments the nucleic acids can be heterogeneous, such thatindividual molecules having different sequences are present at the siteor feature. The nucleic acids are generally covalently attached to thesolid support, for example, via their 5′ ends, but in some cases otherattachment means are possible. The nucleic acids in a cluster can besingle stranded or double stranded. In some but not all embodiments,clusters are made by a solid-phase amplification method known as bridgeamplification. Exemplary configurations for clusters and methods fortheir production are set forth, for example, in U.S. Pat. No. 5,641,658;U.S. Patent Publ. No. 2002/0055100; U.S. Pat. No. 7,115,400; U.S. PatentPubl. No. 2004/0096853; U.S. Patent Publ. No. 2004/0002090; U.S. PatentPubl. No. 2007/0128624; and U.S. Patent Publ. No. 2008/0009420, each ofwhich is incorporated herein by reference.

As used herein, the term “deblocking agent” means a catalyst, enzyme,reagent or other substance that is capable of modifying or removing ablocking moiety. In particular embodiments, a deblocking agent can havespecificity for a particular blocking moiety. As such the deblockingagent may selectively remove a particular blocking moiety from anucleotide analog compared to another blocking moiety. Exemplarydeblocking agents include, but are not limited to, an enzyme, such as aphosphoesterase, esterase, alkyl transferase or methyl transferase; or achemical reagent such as a phosphine, proton, or chemical catalyst, suchas palladium catalyst, or the like. Further examples of deblockingagents are set forth in further detail below.

As used herein, the term “different”, when used in reference to nucleicacids, means that the nucleic acids have nucleotide sequences that arenot the same as each other. Two or more nucleic acids can havenucleotide sequences that are different along their entire length.Alternatively, two or more nucleic acids can have nucleotide sequencesthat are different along a substantial portion of their length. Forexample, two or more nucleic acids can have target nucleotide sequenceportions that are different from each other while also having auniversal sequence region that is the same for both. Generally, when twospecies are referred to herein as being “different,” one of the specieswill have a structural property that is not the same as the structuralproperties of the second species. For example, two different polymericspecies (such as two proteins) can have different sequences of monomericsubunits (such as different sequences of amino acids for two differentproteins).

As used herein, the term “each,” when used in reference to a collectionof items, is intended to identify an individual item in the collectionbut does not necessarily refer to every item in the collection.Exceptions can occur if explicit disclosure or context clearly dictatesotherwise.

As used herein, the term “nucleic acid” is intended to be consistentwith its use in the art and includes naturally occurring nucleic acidsor functional analogs thereof. Particularly useful functional analogsare capable of hybridizing to a nucleic acid in a sequence specificfashion or capable of being used as a template for replication of aparticular nucleotide sequence. Naturally occurring nucleic acidsgenerally have a backbone containing phosphodiester bonds. An analogstructure can have an alternate backbone linkage including any of avariety of those known in the art. Naturally occurring nucleic acidsgenerally have a deoxyribose sugar (e.g. found in deoxyribonucleic acid(DNA)) or a ribose sugar (e.g. found in ribonucleic acid (RNA)). Anucleic acid can contain any of a variety of analogs of these sugarmoieties that are known in the art. A nucleic acid can include native ornon-native bases. In this regard, a native deoxyribonucleic acid canhave one or more bases selected from the group consisting of adenine,thymine, cytosine or guanine and a ribonucleic acid can have one or morebases selected from the group consisting of uracil, adenine, cytosine orguanine. Useful non-native bases that can be included in a nucleic acidare known in the art. The term “target,” when used in reference to anucleic acid, is intended as a semantic identifier for the nucleic acidin the context of a method or composition set forth herein and does notnecessarily limit the structure or function of the nucleic acid beyondwhat is otherwise explicitly indicated.

As used herein, the term “nucleic acid template” refers to a nucleicacid or portion thereof that is capable of use as a guide for polymerasecatalyzed replication. A nucleic acid molecule can include multipletemplates along its length or, alternatively, only a single template permolecule may be used in a particular embodiment herein. A nucleic acidtemplate can also function as a guide for ligase-catalyzed primerextension.

As used herein, the term “nucleotide” or “nucleotide analog” is intendedto include natural nucleotides, non-natural nucleotides,ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides and othermolecules known as nucleotides. For example, the terms are used hereinto generally refer to a nucleoside moiety (whether ribose, deoxyribose,or analog thereof) including a base moiety and optionally attached toone or more phosphate moieties. The term can be used to refer to amonomer unit that is present in a polymer, for example, to identify asubunit present in a DNA or RNA strand. The term can also be used torefer to a monomeric molecule that is not necessarily present in apolymer, for example, a molecule that is capable of being incorporatedinto a polynucleotide in a template dependent manner by a polymerase.

Exemplary nucleotide analogs include, but are not limited to,ribonucleotide monophosphate (sometimes referred to as a ribonucleosidemonophosphate), ribonucleotide diphosphate (sometimes referred to as aribonucleoside diphosphate), ribonucleotide triphosphate (sometimesreferred to as a ribonucleoside triphosphate), deoxynucleotidemonophosphate (sometimes referred to as a deoxynucleosidemonophosphate), deoxynucleotide diphosphate (sometimes referred to as adeoxynucleoside diphosphate) and deoxynucleotide triphosphate (sometimesreferred to as a deoxynucleoside triphosphate). For clarity when wishingto distinguish RNA components from DNA components, the term“ribonucleotide” can be used to specify RNA nucleotides, such asribouridine triphosphate, riboguanidine triphosphate, ribocytidinetriphosphate or riboadenosine triphosphate; and the term“deoxynucleotide” can be used to specify DNA nucleotides, such asdeoxythymidine triphosphate, deoxyguanidine triphosphate, deoxycytidinetriphosphate and deoxyadenosine triphosphate. In particular embodiments,the nucleotides are ‘extendable’, for example, lacking an extensionblocking moiety at the 3′ hydroxyl or at any other position on thenucleotide. In other embodiments, the nucleotides are ‘blocked,’ havinga moiety that prevents the 3′ position from participating in extensionby addition of another nucleotide or oligonucleotide.

As used herein, the term “pitch” refers to the center to center distancefor two sites in an array. A pattern of sites can be regular such thatthe coefficient of variation around the average pitch is small or thepattern can be non-regular in which case the coefficient of variationcan be relatively large. In either case, the average pitch can be, forexample, at least 10 nm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 100 μm ormore. Alternatively or additionally, the average pitch can be, forexample, at most 100 μm, 10 μm, 5 μm, 1 μm, 0.5 μm 0.1 μm or less. Ofcourse, the average pitch for a particular pattern of sites can bebetween one of the lower values and one of the upper values selectedfrom the ranges above.

As used herein, the term “primer” means a nucleic acid having a sequencethat binds to a primer binding site at or near a template sequence.Generally, the primer binds in a configuration that allows replicationof the template, for example, via polymerase extension of the primer.The primer can be a first portion of a nucleic acid molecule that bindsto a second portion of the nucleic acid molecule, the first portionbeing a primer sequence and the second portion being a primer bindingsequence (e.g. a hairpin primer). Alternatively, the primer can be afirst nucleic acid molecule that binds to a second nucleic acid moleculehaving the template sequence. A primer can consist of DNA, RNA oranalogs thereof.

As used herein, the term “primer extension product” means a primer thathas been modified by addition of at least one nucleotide analog. Forexample, a primer can be modified by addition of one or more nucleotideanalogs to its 3′ end (e.g. via polymerase catalysis), thereby forming aprimer extension product. A primer extension product can alternativelybe produced by ligation of an oligonucleotide to the 3′ or 5′ end of aprimer. In this case, the primer extension product is extended by alength equivalent to the length of the oligonucleotide. A primerextension product can be at least 1, 2, 5, 10, 500, 1000 or morenucleotides longer than the primer. Alternatively or additionally, aprimer extension product can be no more than 1, 2, 5, 10, 500, or 1000nucleotides longer than the primer. For example, use of a blockednucleotide analog provides for an extension product that is at least 1nucleotide longer than the primer and also no more than 1 nucleotidelonger than the primer.

As used herein, reference to “selectively” manipulating (or “selective”manipulation of) a first thing compared to second thing is intended tomean that the manipulation has a greater effect on the first thingcompared to the effect on the second thing. The manipulation need nothave any effect on the second thing. The manipulation can have an effecton the first thing that is at least 1%, 5%, 10%, 25%, 50%, 75%, 90%,95%, or 99% greater than the effect on the second thing. Themanipulation can have an effect on the first thing that is at least 2fold, 3 fold, 4 fold, 5 fold, 10 fold, 100 fold, 1×10³ fold, 1×10⁴ foldor 1×10⁶ fold higher than the effect on the second thing. Themanipulation can include, for example, modifying, contacting, treating,changing, cleaving (e.g. of a chemical bond), photo-chemically cleaving(e.g. of a chemical bond), forming (e.g. of a chemical bond),photo-chemically forming (e.g. of a chemical bond), covalentlymodifying, non-covalently modifying, destroying, photo-ablating,removing, synthesizing, polymerizing, photo-polymerizing, amplifying(e.g. of a nucleic acid), copying (e.g. of a nucleic acid), extending(e.g. of a nucleic acid), ligating (e.g. of a nucleic acid), or othermanipulation set forth herein or otherwise known in the art.

As used herein, the term “sequence,” when used in reference to a nucleicacid, refers to the order of nucleotides (or bases) in the nucleicacids. In cases where, different species of nucleotides are present inthe nucleic acid, the sequence includes an identification of the speciesof nucleotide (or base) at respective positions in the nucleic acid. Asequence is a property of all or part of a nucleic acid molecule. Theterm can be used similarly to describe the order and positional identityof monomeric units in other polymers such as amino acid monomeric unitsof protein polymers.

As used herein, the term “site” means a location in an array where atleast one analyte molecule is present. A site can contain only a singleanalyte molecule or it can contain a population of several analytemolecules of the same species. In some embodiments, a site can includemultiple different analyte molecule species, each species being presentin one or more copies. Sites of an array are typically discrete. Thediscrete sites can be contiguous or they can have spaces between eachother.

As used herein, the term “species” or “type” is used to identifymolecules that share the same chemical structure. For example, a mixtureof nucleotide analogs can include several dCTP molecules. The dCTPmolecules will be understood to be the same species, or type, as eachother, but a different species, or types, compared to dATP, dGTP, dTTPetc. Similarly, individual DNA molecules that have the same sequence ofnucleotides are the same species, or type, whereas DNA molecules withdifferent sequences are different species or types. As another example,a DNA polymerase is a different polymerase species, or type, compared toan RNA polymerase (even if the two polymerases are derived from the sameorganism).

As used herein, the term “universal sequence” refers to a sequence thatis common to two or more nucleic acid molecules, even where themolecules also have other regions of sequence that differ from eachother. A universal sequence that is present in different members of acollection of molecules can allow capture of multiple different nucleicacids using a population of universal capture nucleic acids that arecomplementary to the universal sequence. Similarly, a universal sequencepresent in different members of a collection of molecules can allow thereplication or amplification of multiple different nucleic acids using apopulation of universal primers that are complementary to the universalsequence. Thus a universal capture nucleic acid or a universal primerincludes a sequence that can hybridize specifically to a universalsequence. Target nucleic acid molecules may be modified to attachuniversal adapters, for example, at one or both ends of the differenttarget sequences.

The embodiments set forth below and recited in the claims can beunderstood in view of the above definitions.

The present disclosure provides a method for sequencing nucleic acidtemplates. The method can include steps of (a) providing an array ofsites, wherein each site comprises a mixture of at least two differentnucleic acid templates; (b) extending primers hybridized to thedifferent nucleic acid templates at each of the sites with differentnucleotide analogs having different reversible blocking moieties,respectively, thereby producing different primer extension products ateach site; (c) detecting the different primer extension products todistinguish the different nucleotide analogs at each site; (d) removingthe different reversible blocking moieties from the primer extensionproducts at each of the sites using a first treatment that is selectivefor a first of the different reversible blocking moieties and a secondtreatment that is selective for a second of the different reversibleblocking moieties; and (e) repeating (b) through (d) to determine thesequence of different nucleotide analogs added to each of the differentextension products at each of the sites.

As set forth above, a method of the present disclosure can include astep of providing first and second nucleic acid templates, wherein thesequences for the two templates are different. The two templatesequences can be portions of a single nucleic acid molecule or,alternatively, the two template sequences can be located on separatemolecules. As set forth in further detail elsewhere herein, the twotemplate sequences may be in a proximity that is too close to spatiallyresolve with the detection system used. Nevertheless, the orthogonaldetection methods of the present disclosure allow these templatesequences to be distinguished. The orthogonal detection scheme isexemplified for two template sequences, but can be used with two or moretemplate sequences. Accordingly, a system or method set forth herein caninclude at least 2, 3, 4, 5, 10 or more template sequences that are inclose proximity, for example on a single nucleic acid molecule, at asingle site of an array, or otherwise in a proximity that is too closeto spatially resolve with the detection system used.

Target nucleic acids used herein can be composed of DNA, RNA or analogsthereof. The source of the target nucleic acids can be genomic DNA,messenger RNA, or other nucleic acids from native sources. In some casesthe target nucleic acids that are derived from such sources can beamplified prior to use in a method or composition herein.

Exemplary biological samples from which target nucleic acids can bederived include, for example, those from a mammal such as a rodent,mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow,cat, dog, primate, human or non-human primate; a plant such asArabidopsis thaliana, corn, sorghum, oat, wheat, rice, canola, orsoybean; an algae such as Chlamydomonas reinhardtii; a nematode such asCaenorhabditis elegans; an insect such as Drosophila melanogaster,mosquito, fruit fly, honey bee or spider; a fish such as zebrafish; areptile; an amphibian such as a frog or Xenopus laevis; a Dictyosteliumdiscoideum; a fungi such as Pneumocystis carinii, Takifugu rubripes,yeast, Saccharomyces cerevisiae or Schizosaccharomyces pombe; or aPlasmodium falciparum. Target nucleic acids can also be derived from aprokaryote such as a bacterium, Escherichia coli, staphylococci orMycoplasma pneumoniae; an archae; a virus such as Hepatitis C virus orhuman immunodeficiency virus; or a viroid. Target nucleic acids can bederived from a homogeneous culture or population of the above organismsor alternatively from a collection of several different organisms, forexample, in a community or ecosystem. Nucleic acids can be isolatedusing methods known in the art including, for example, those describedin Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition,Cold Spring Harbor Laboratory, New York (2001) or in Ausubel et al.,Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore,Md. (1998), each of which is incorporated herein by reference.

In particular embodiments, a nucleic acid sample can be modified orprepared for use in one or more of the methods set forth herein. In somecases it is desired to add one or more primer binding sites to a nucleicacid. Known molecular biological techniques can be used to introduceprimer binding sites upstream of respective template sequences, forexample, via insertion of an adapter having the primer binding site,mutation to create the primer binding site, ligation of an adapterhaving the primer binding site etc. Useful methods are described inSambrook et al., supra and Ausubel et al., supra. US Pat. App. Pub. No.2015/0031560 A1 (which is incorporated herein by reference) provides anillustration of a tagmentation-based technique for introducing primerbinding sites. Tagmentation is particularly useful for introducing oneor more primer binding sites and can be carried out, for example, usingtechniques set forth in U.S. Pat. Nos. 6,294,385 and 8,383,345, and PCTPub. No. WO 2012/106546, each of which is incorporated herein byreference. It will be understood that in some cases naturally occurringsequence regions that reside upstream of respective template sequencescan be exploited as primer binding sites in a method set forth herein.Methods similar to those exemplified above for primer binding sites canbe used to introduce other desired sequence elements such as promoterelements for RNA polymerase-based extension or tag sequences.

Universal priming sites are particularly useful for multiplexapplications of the methods set forth herein. Universal priming sitesprovide a region of sequence that is common to two or more nucleic acidmolecules where the molecules also have template or target regions withdifferent sequences. A universal priming sequence present in differentmembers of a collection of molecules can allow the replication,amplification or detection of multiple different sequences using asingle universal primer species that is complementary to the universalpriming sequence. Thus, a universal primer includes a sequence that canhybridize specifically to a universal priming sequence. Examples ofmethods of attaching universal sequences to a collection of targetnucleic acids can be found in US Pat. App. Pub. No. 2007/0128624 A1,which is incorporated herein by reference.

In some embodiments, target nucleic acids can be obtained as fragmentsof one or more larger nucleic acids. Fragmentation can be carried outusing any of a variety of techniques known in the art including, forexample, nebulization, sonication, chemical cleavage, enzymaticcleavage, or physical shearing. Fragmentation may also result from useof a particular amplification technique that produces amplicons bycopying only a portion of a larger nucleic acid. For example, PCRamplification produces fragments having a size defined by the length ofthe fragment between the flanking primers used for amplification.

A population of target nucleic acids, or amplicons thereof, can have anaverage strand length that is desired or appropriate for a particularapplication of the methods or compositions set forth herein. Forexample, the average strand length can be less than about 100,000nucleotides, 50,000 nucleotides, 10,000 nucleotides, 5,000 nucleotides,1,000 nucleotides, 500 nucleotides, 100 nucleotides, or 50 nucleotides.Alternatively or additionally, the average strand length can be greaterthan about 10 nucleotides, 50 nucleotides, 100 nucleotides, 500nucleotides, 1,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides,50,000 nucleotides, or 100,000 nucleotides. The average strand lengthfor a population of target nucleic acids, or amplicons thereof, can bein a range between a maximum and minimum value set forth above. It willbe understood that amplicons generated at an amplification site (orotherwise made or used herein) can have an average strand length that isin a range between an upper and lower limit selected from thoseexemplified above.

In some cases a population of target nucleic acids can be produced orotherwise configured to have a maximum length for its members. Forexample, the maximum length for the members that are made or used as setforth herein can be less than about 100,000 nucleotides, 50,000nucleotides, 10,000 nucleotides, 5,000 nucleotides, 1,000 nucleotides,500 nucleotides, 100 nucleotides or 50 nucleotides. Alternatively oradditionally, a population of target nucleic acids, or ampliconsthereof, can be produced under conditions or otherwise configured tohave a minimum length for its members. For example, the minimum lengthfor the members that are used in one or more steps of a method set forthherein or that are present in a particular composition can be more thanabout 10 nucleotides, 50 nucleotides, 100 nucleotides, 500 nucleotides,1,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, 50,000nucleotides, or 100,000 nucleotides. The maximum and minimum strandlength for target nucleic acids in a population can be in a rangebetween a maximum and minimum value set forth above. It will beunderstood that amplicons generated at a site of an array (or otherwisemade or used herein) can have maximum and/or minimum strand lengths in arange between the upper and lower limits exemplified above.

Any of a variety of known amplification techniques can be used toincrease the amount of template sequences present for use in a methodset forth herein. Exemplary techniques include, but are not limited to,polymerase chain reaction (PCR), rolling circle amplification (RCA),multiple displacement amplification (MDA), or random prime amplification(RPA) of nucleic acid molecules having template sequences. It will beunderstood that amplification of target nucleic acids prior to use in amethod or composition set forth herein is optional. As such, targetnucleic acids will not be amplified prior to use in some embodiments ofthe methods and compositions set forth herein. Target nucleic acids canoptionally be derived from synthetic libraries. Synthetic nucleic acidscan have native DNA or RNA compositions or can be analogs thereof.Solid-phase amplification methods can also be used, including forexample, cluster amplification, bridge amplification or other methodsset forth below in the context of array-based methods.

A nucleic acid used in a method set forth herein can be solution phaseor solid-phase. The nucleic acid when in solution phase is generallysoluble, but can also be in a suspended form that is capable of beingprecipitated, as is the case for some large nucleic acid species such aschromosomes or nucleic acid nanoballs (see, for example, US Pat. App.Pub. No. 2007/0099208 A1, which is incorporated herein by reference). Anucleic acid that is solid-phase can occur in or on a solid-phasesupport. Exemplary solid-phase supports include those made from glass,nitrocellulose, silica, metal, plastic and other materials set forthelsewhere herein, for example, with regard to array formats and flowcells. Similarly, a nucleic acid can occur in or on a semisolid supportsuch as a gel. Exemplary gels that are useful include, but are notlimited to, those having a colloidal structure, such as agarose; polymermesh structure, such as gelatin; or cross-linked polymer structure, suchas polyacrylamide. Hydrogels are particularly useful such as those setforth in US Pat. App. Pub. No. 2011/0059865 A1 and U.S. Pat. No.9,012,022, each of which is incorporated herein by reference.

Attachment of a nucleic acid to a support, whether rigid or semi-rigid,can occur via covalent or non-covalent linkage(s). Exemplary linkagesare set forth in U.S. Pat. Nos. 6,737,236; 7,259,258; 7,375,234 and7,427,678; and US Pat. App. Pub. No. 2011/0059865 A1, each of which isincorporated herein by reference. In some embodiments, a nucleic acid orother reaction component can be attached to a gel or other semisolidsupport that is in turn attached or adhered to a solid-phase support. Insuch embodiments, the nucleic acid or other reaction component will beunderstood to be solid-phase.

A multiplex reaction can utilize a solid-phase support (a.k.a. a solidsupport). A solid-phase support can be useful for separating individualreactions such that each can be interrogated separately or individually.For example, several different nucleic acids in a mixture can beattached to the solid-phase support. The nucleic acids can be attachedto the solid-phase support in an array format.

In some embodiments, an array of sites is provided, wherein each siteincludes a first nucleic acid template and a second nucleic acidtemplate and wherein the first nucleic acid template has a sequence thatis different from the sequence of the second nucleic acid template.There can be greater than two different templates per site in someembodiments. Exemplary array materials and manufacturing methods thatcan be modified for use herein include, without limitation, a BeadChipArray available from Illumina®, Inc. (San Diego, Calif.) or arrays suchas those described in U.S. Pat. Nos. 6,266,459, 6,355,431, 6,770,441,6,859,570 or 7,622,294; or PCT Pub. No. WO 00/63437, each of which isincorporated herein by reference. Further examples of commerciallyavailable arrays that can be used include, for example, an Affymetrix®GeneChip® array or other array synthesized in accordance with techniquessometimes referred to as VLSIPS™ (Very Large Scale Immobilized PolymerSynthesis) technologies. A spotted array can also be used according tosome embodiments. An exemplary spotted array is a CodeLink™ Arrayavailable from Amersham Biosciences. Another array that is useful is onethat is manufactured using inkjet printing methods such as SurePrint™Technology available from Agilent Technologies.

Other useful arrays that can be used, for example, by modifying thesites to include multiple different template nucleic acid sequences,include those that are used in nucleic acid sequencing applications. Forexample, arrays having amplicons of genomic fragments (often referred toas clusters) are particularly useful such as those described in Bentleyet al., Nature 456:53-59 (2008), PCT Pub. Nos. WO 04/018497, WO91/06678, or WO 07/123744; U.S. Pat. Nos. 7,057,026, 7,329,492,7,211,414, 7,315,019, or 7,405,281; or US Pat. App. Pub. No.2008/0108082 A1, each of which is incorporated herein by reference.

Nucleic acid clusters can be created by solid-phase amplificationmethods. For example, a nucleic acid having one or more templatesequences to be detected can be attached to a surface and amplifiedusing bridge amplification. Useful bridge amplification methods aredescribed, for example, in U.S. Pat. Nos. 5,641,658 or 7,115,400; orU.S. Pat. App. Pub. Nos. 2002/0055100 A1, 2004/0096853 A1, 2004/0002090A1, 2007/0128624 A1 or 2008/0009420 A1, each of which is incorporatedherein by reference. Another useful method for amplifying nucleic acidson a surface is rolling circle amplification (RCA), for example, asdescribed in Lizardi et al., Nat. Genet. 19:225-232 (1998) and US Pat.App. Pub. No. 2007/0099208 A1, each of which is incorporated herein byreference. Another type of array that is useful is an array of particlesproduced from an emulsion PCR amplification technique. Examples aredescribed in Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822(2003), PCT app. Pub. No. WO 05/010145, or US Pat App. Pub. No.2005/0130173 A1 or 2005/0064460 A1, each of which is incorporated hereinby reference. Although the above arrays have been described in thecontext of sequencing applications, it will be understood that thearrays can be used in other embodiments including, for example, thosethat use a non-cyclic primer extension technique.

Detection can be carried out at ensemble or single molecule levels on anarray. Ensemble level detection is detection that occurs in a way thatseveral copies of a single template sequence (e.g. multiple amplicons ofa template) are detected at each individual site and individual copiesat the site are not distinguished from each other. Thus, ensembledetection provides an average signal from many copies of a particulartemplate sequence at the site. For example, the site can contain atleast 10, 100, 1000 or more copies of a particular template sequence. Ofcourse, a site can contain multiple different template sequences each ofwhich is present as an ensemble. Alternatively, detection at a singlemolecule level includes detection that occurs in a way that individualtemplate sequences are individually resolved on the array, each at adifferent site. Thus, single molecule detection provides a signal froman individual molecule that is distinguished from one or more signalsthat may arise from a population of molecules within which theindividual molecule is present. Of course, even in a single moleculearray, a site can contain several different template sequences (e.g. twoor more template sequence regions located along a single nucleic acidmolecule).

An array of sites can appear as a grid of spots or patches. The sitescan be located in a repeating pattern or in an irregular non-repeatingpattern. Particularly useful patterns are hexagonal patterns,rectilinear patterns, grid patterns, patterns having reflectivesymmetry, patterns having rotational symmetry, or the like. Asymmetricpatterns can also be useful.

The size of the sites and/or spacing between the sites in an array canvary to achieve high density, medium density or lower density. Highdensity arrays are characterized as having sites with a pitch that isless than about 15 μm. Medium density arrays have sites with a pitchthat is about 15 to 30 μm, while low density arrays have a pitch that isgreater than 30 μm. An array useful in some embodiments can have siteswith a pitch that is less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5μm. An embodiment of the methods set forth herein can be used to imagean array at a resolution sufficient to distinguish sites at the abovedensities or density ranges. However, the detecting step will typicallyuse a detector having a spatial resolution that is too low to resolvepoints at distance equivalent to the spacing between a first template(or first primer extension product hybridized thereto) and a secondtemplate (or second primer extension product hybridized thereto) at anindividual site. In particular embodiments, sites of an array can eachhave an area that is larger than about 100 nm², 250 nm², 500 nm², 1 μm²,2.5 μm², 5 μm², 10 μm², 100 μm², or 500 μm². Alternatively oradditionally, sites of an array can each have an area that is smallerthan about 1 mm², 500 μm², 100 μm², 25 μm², 10 μm², 5 μm², 1 μm², 500nm², or 100 nm². Indeed, a site can have a size that is in a rangebetween an upper and lower limit selected from those exemplified above.

The methods set forth herein can use arrays having sites at any of avariety of densities including, for example, at least about 10sites/cm², 100 sites/cm², 500 sites/cm², 1,000 sites/cm², 5,000sites/cm², 10,000 sites/cm², 50,000 sites/cm², 100,000 sites/cm²,1,000,000 sites/cm², 5,000,000 sites/cm², or higher.

Generally, an array will have sites with different nucleic acid sequencecontent. Accordingly, each of the sites in an array can contain anucleic acid sequence that is unique compared to the nucleic acidsequences at the other sites in the array. However, in some cases anarray can have redundancy such that two or more sites have the samenucleic acid content.

It will be understood that the steps of the methods set forth herein canbe carried out in a manner to expose an entire site or a plurality ofsites of an array with the treatment. For example, a step that involvesextension of a primer can be carried out by delivering primer extensionreagents to an array such that multiple nucleic acids (e.g. differentnucleic acids in a mixture) at each of one or more sites of the arrayare contacted with the primer extension reagents. Similarly a step ofdeblocking a blocked primer extension product can be carried out byexposing an array with a deblocking treatment such that multiple nucleicacids (e.g. different nucleic acids in a mixture) at each of one or moresites of the array are contacted with the treatment.

At any given point in a sequencing-by-synthesis, or other primerextension reaction, the species of nucleotide that is present in a firsttemplate at the position that complements the site of primer extensioncan be the same as the species of nucleotide that is present in a secondtemplate at the position that complements the site of primer extension.In other words, the first nucleic acid template at a particular site ofan array can include at least one base moiety that is the same speciesas a base moiety in the second nucleic acid template at that particularsite, and a complementary nucleotide analog can be added to each of theprimers at the positions in the templates where those base moietiesreside. This can be the case whether or not the template sequences towhich the primers are hybridized are the same or different. Techniquesset forth in further detail below can be used to distinguish the twotemplates, for example, the use of different sets of nucleotide analogshaving mutually distinguishable labels.

Any of a variety of polymerases can be used in a method or compositionset forth herein including, for example, protein-based enzymes isolatedfrom biological systems and functional variants thereof. Reference to aparticular polymerase, such as those exemplified below, will beunderstood to include functional variants thereof unless indicatedotherwise. A particularly useful function of a polymerase is to catalyzethe polymerization of a nucleic acid strand using an existing nucleicacid as a template. Other functions that are useful are describedelsewhere herein. Examples of useful polymerases include DNApolymerases, reverse transcriptases and RNA polymerases.

A polymerase having an intrinsic 3′ to 5′ proofreading exonucleaseactivity can be useful for some embodiments. Polymerases thatsubstantially lack 3′ to 5′ proofreading exonuclease activity are alsouseful in some embodiments, for example, in most sequencing embodiments.Absence of exonuclease activity can be a wild type characteristic or acharacteristic imparted by a variant or engineered polymerase structure.For example, exo minus Klenow fragment is a mutated version of Klenowfragment that lacks 3′ to 5′ proofreading exonuclease activity.

Depending on the embodiment that is to be used, a polymerase can beeither thermophilic or heat inactivatable. Thermophilic polymerases aretypically useful for high temperature conditions or in thermocyclingconditions such as those employed for polymerase chain reaction (PCR)techniques. Examples of thermophilic polymerases include, but are notlimited to 9° N DNA Polymerase, Taq DNA polymerase, Phusion® DNApolymerase, Pfu DNA polymerase, RB69 DNA polymerase, KOD DNA polymerase,and VentR® DNA polymerase. Most polymerases isolated fromnon-thermophilic organisms are heat inactivatable. Examples are DNApolymerases from phage. It will be understood that polymerases from anyof a variety of sources can be modified to increase or decrease theirtolerance to high temperature conditions. Particularly usefulpolymerases for incorporating nucleotide analogs having labels and/orreversible terminating moieties are described in US 2006/0281109 A1,which is incorporated herein by reference.

In particular embodiments of the methods and compositions set forthherein, only a single species of polymerase will be used. In suchexamples, each site of an array will interact with only one species ofpolymerase at a given time even though multiple polymerases may bepresent at the site and bound to multiple primed-templates at each site.For example, all sites of an array may interact with a particularspecies of DNA polymerase and other polymerases (such as RNApolymerases) will be absent from the array.

Another extension technique that can be modified for use in a method orcomposition set forth herein is a ligase based system that is selectivefor incorporation of oligonucleotides instead of monomeric nucleotidesthat are incorporated by the polymerase-based extension systemsdescribed above. A DNA ligase reagent system that uses a first set ofoligonucleotides having a reversible blocking moiety that is selectivelydeblocked by a first deblocking treatment is orthogonal with a reagentsystem that uses a second set of oligonucleotides having a reversibleblocking moiety that is selectively deblocked by a second deblockingtreatment. Deblocking of primers extended with reversibly blockedoligonucleotides can be carried out in an orthogonal fashion much likeexemplified herein for deblocking of primers extended with reversiblyblocked nucleotide analogs. Extension by ligation can be carried out ina sequencing application using a population of partially random probeoligonucleotides having a one- or two-base encoding scheme. Ligationbased extension techniques that can be modified for use herein, such asin a sequencing context, are set forth in McKernan et al., GenomeResearch 19 (9): 1527-41 (2009); Shendure et al., Science 309:1728-1732(2005); or U.S. Pat. Nos. 5,599,675 or 5,750,341, each of which isincorporated herein by reference.

A nucleic acid extension reaction, or other cyclic reaction, that iscarried out using methods set forth herein can proceed for one or morecycles. In particular embodiments, a multicycle reaction can include atleast 2 cycles, 5 cycles, 10 cycles, 50 cycles, 100 cycles, 500 cycles,1,000 cycles, 5,000 cycles, 10,000 cycles or more. Alternatively oradditionally, a reaction can have an upper limit whereby no more than 1cycle, 2 cycles, 5 cycles, 10 cycles, 50 cycles, 100 cycles, 500 cycles,1,000 cycles, 5,000 cycles, or 10,000 cycles occur. In some embodiments,each cycle will result in the incorporation of a single nucleotideanalog into an extended primer. In this case, the minimum or maximumnumber of cycles exemplified above can be understood to exemplify theminimum or maximum number of nucleotides incorporated into an extensionproduct in a polymerase catalyzed reaction.

Some embodiments can use non-cyclic extension reactions such as singlebase extension (SBE) or allele specific primer extension (ASPE)reactions. Reversible terminator moieties can be used to achieveorthogonal extension of two different primers in a non-cyclic extensionformat. Since a deblocking step is not necessary for continuation ofthese non-cyclic reactions (i.e. once the primers have been extended),the nucleotide analogs used in the extension step can instead benon-reversibly terminated. For example, dideoxynucleotides can be used.Exemplary reagents and related techniques for SBE, ASPE and other usefulnon-cyclic extension techniques are described, for example, in U.S. Pat.No. 7,670,810 or U.S. Pat. App. Pub. Nos. 2003/0108867; 2003/0108900;2003/0170684; 2003/0207295; or 2005/0181394, each of which isincorporated herein by reference. An example of a commercially availableproduct that uses a non-cyclic extension technique and that can bemodified to increase information content via the orthogonal detectionmethods set forth herein is the Infinium® genotyping product availablefrom Illumina, Inc. (San Diego, Calif.).

Cyclic and non-cyclic reactions alike can include steps where reactioncomponents are separated from each other or removed from the reactionenvironment. One or more reaction components can be separated, forexample, by separation of solid-phase components from liquid-phasecomponents. Wash steps can optionally be included in order to morecompletely remove unwanted liquid-phase component(s) from solid-phasecomponent(s). A particularly useful reaction vessel for such separationsis a flow cell such as those commonly used in cyclical sequencingprocedures. Exemplary flow cells, methods for their manufacture andmethods for their use are described in US Pat. App. Publ. Nos.2010/0111768 A1 or 2012/0270305 A1; or PCT App. Pub. No. WO 05/065814,each of which is incorporated herein by reference. Whether or notsolid-phase separation methods are used, reaction components can beremoved by any of a variety of other techniques known in the artincluding, liquid-liquid extraction, solid-phase extraction,chromatography, filtration, centrifugation or the like.

Reversible terminator moieties provide a convenient way to control anextension reaction to add only a single nucleotide to a primer until asubsequent deblocking step is carried out. As set forth herein, the useof orthogonal blocking moieties and deblocking treatments provide superresolution detection, whereby a greater complexity of templates can bemonitored and detected than would otherwise be allowed using anon-orthogonal technique. Examples of reversible blocking moieties andtheir deblocking conditions include, but are not limited to, moietiesthat can be photocleavably removed from the 3′ position such aso-nitrobenzylethers and alkyl o-nitrobenzyl carbonate; ester moietiesthat can be removed by base hydrolysis; allyl-moieties that can beremoved with NaI, chlorotrimethylsilane and Na₂S₂O₃ or with Hg(II) inacetone/water; -azidomethyl (—CH₂—N₃) which can be cleaved withphosphines, such as tris(2-carboxyethyl)phosphine (TCEP) ortri(hydroxypropyl)phosphine (THP); acetals, such as tert-butoxy-ethoxywhich can be cleaved with acidic conditions; MOM (—CH₂OCH₃) moietiesthat can be cleaved with LiBF₄ and CH₃CN/H₂O; 2,4-dinitrobenzenesulfenyl which can be cleaved with nucleophiles such as thiophenol andthiosulfate; tetrahydrofuranyl ether which can be cleaved with Ag(I) orHg(II); and 3′ phosphate which can be cleaved by phosphatase enzymes(e.g. polynucleotide kinase). Other useful reversible moieties includeethers, —F, —NH₂, —OCH₃, —N₃, —NHCOCH₃, and 2-nitrobenzene carbonate.Useful deblocking treatments include irradiation with light (e.g. toinduce photocleavage), heating, exposure to chemical reactants, exposureto catalysts, exposure to electrical current (e.g. to induceelectrolysis) or the like.

Particular embodiments of the methods herein can employ reversiblyblocked primers and reversibly blocked nucleotide analogs, for example,in a multicycle process such as sequencing-by-synthesis. As exemplifiedpreviously herein, a particular reversibly blocked primer and aparticular set of reversibly blocked nucleotide analogs can besusceptible to the same deblocking conditions. This can be due to thesame species of reversible blocking moiety being present on the primerand on the nucleotide analogs in the set. However, it is also possibleto use different blocking moieties that are nonetheless susceptible tothe same deblocking treatment.

A reversible blocking moiety can be attached to the 3′ nucleotide of aprimer. Generally, the reversible blocking moiety is attached at the 3′positon of the ribose sugar moiety. However, a blocking moiety can beattached to alternative positions instead, including for example, at the2′ position of the ribose or on the base moiety. A blocking moiety canalso be attached at the 5′ end of a primer, for example, at the 5′position of the ribose of the terminal nucleotide or at the 5′ phosphatemoiety. Primers that are blocked at the 5′ end can be useful forembodiments that employ ligation techniques.

The 3′ or 5′ end of a primer can be attached to a label moiety such asan optical label. The label can be present whether or not a reversibleblocking moiety is also present on the primer. In some cases, aparticular moiety can function as both a label and as a reversible blockto primer extension.

The same attachment points for a label and/or reversible blocking moietythat are exemplified above for primers, can be useful for individualnucleotides.

Further exemplary guidance regarding blocking moieties and deblockingtreatments are described, for example, in Bentley et al., Nature456:53-59 (2008), PCT App. Pub. Nos. WO 04/018497, WO 91/06678 or WO07/123744; U.S. Pat. Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019,8,088,575 or 7,405,281; or US Pat. App. Pub. No. 2008/0108082 A1, eachof which is incorporated herein by reference.

Orthogonal manipulation and detection in accordance with the presentdisclosure does not require that two template sequences differ at everyposition along their length. Rather, the same base moiety can be presentat positions that are detected on a first template and second template,respectively. The two positions can be distinguished based on thedistinguishable characteristics of the labels present in the orthogonalreagent systems and the specificity of the reagent systems for extendingthe appropriately deblocked primer. This information can in turn be usedto distinguishably detect the two different template sequences, even ifthe two positions are detected simultaneously using a detector having aresolution that is too low to resolve points at distance equivalent tothe spacing of the two template sequences.

Any of a variety of labels can be used. A label moiety that isparticularly useful when used for detection of a nucleotide analog, canbe any part of the nucleotide analog that provides a distinguishablecharacteristic when compared to other molecules present in itsenvironment. The distinguishable characteristic can be, for example, anoptical signal such as absorbance of radiation, fluorescence emission,luminescence emission, fluorescence lifetime, fluorescence polarization,or the like; binding affinity for a ligand or receptor; magneticproperties; electrical properties; charge; mass; radioactivity or thelike. Exemplary label moieties include, without limitation, afluorophore, luminophore, chromophore, radioactive isotope, mass label,charge label, spin label, receptor, ligand, or the like. The labelmoiety can be part of a nucleotide that is a monomer unit present in anucleic acid polymer or the label moiety can be a part of a freenucleotide analog (e.g. a nucleotide triphosphate).

Fluorophores are particularly useful and include, for example,fluorescent nanocrystals; quantum dots, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, Cy3, Cy5, stilbene, Lucifer Yellow, CascadeBlue, Texas Red, Alexa dyes, SETA dyes, Atto dyes, phycoerythin, bodipy,and analogs thereof. Useful optical probes are described in Lakowicz,Principles of Fluorescence Spectroscopy, 3^(rd) Ed. Springer (2006);Haugland, Handbook of Fluorescent Probes and Research Products 9^(th)Ed., Molecular Probes, Inc, (2002); Shapiro, Practical Flow Cytometry,4^(th) Ed., John Wiley & Sons (2003); PCT Pat. App. Pub. Nos. WO98/59066 or WO 91/06678; or US Pat. App. Pub. No. 2010/0092957 A1, eachof which is incorporated herein by reference.

Other labels, some of which are non-optical labels, can be used invarious embodiments of the methods and compositions set forth herein.Examples include, without limitation, an isotopic label such as anaturally non-abundant radioactive or heavy isotope; magnetic substance;electron-rich material such as a metal; electrochemiluminescent labelsuch as Ru(bpy)³²⁺; or moiety that can be detected based on a nuclearmagnetic, paramagnetic, electrical, charge to mass, or thermalcharacteristic. Labels can also include magnetic particles or opticallyencoded nanoparticles. Such labels can be detected using appropriatemethods known to those skilled in the art. For example, a charged labelcan be detected using an electrical detector such as those used incommercially available sequencing systems from Ion Torrent (Guilford,Conn., a Life Technologies subsidiary) or detection systems described inUS Pat. App. Publ. Nos. 2009/0026082 A1; 2009/0127589 A1; 2010/0137143A1; or 2010/0282617 A1, each of which is incorporated herein byreference. It will be understood that for some embodiments a nucleotideanalog can be devoid of one or more of the labels set forth herein.

A label moiety can be attached to a nucleotide analog in a variety ofways. Exemplary attachments and label compositions that are useful fornucleotide analogs are set forth in Bentley et al., Nature 456:53-59(2008), PCT App. Pub. Nos. WO 04/018497, WO 91/06678 or WO 07/123744;U.S. Pat. Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019, 8,088,575 or7,405,281; or US Pat. App. Pub. No. 2008/0108082 A1, each of which isincorporated herein by reference.

A detection step of a method set forth herein, can be carried out in amethod of the present disclosure using an apparatus suited to theparticular label in use. For example, an optical detector such as afluorescence detector, absorbance detector, luminescence detector or thelike can be used to detect appropriate optical labels. Systems designedfor array-based detection are particularly useful. For example, opticalsystems for use with the methods set forth herein may be constructed toinclude various components and assemblies as described in U.S. Pat. Nos.8,241,573; 7,329,860 or 8,039,817; or US Pat. App. Pub. Nos.2009/0272914 A1 or 2012/0270305 A1, each of which is incorporated hereinby reference.

An orthogonal detection system, such as a system used forsequencing-by-synthesis, can use different labels to distinguishdifferent nucleotides that are added to each primer. In one embodiment,each nucleotide species will have a unique optical label that produces aunique signal for distinguishing that nucleotide species. An example isan 8 dye approach. In this example, a first set of 4 differentnucleotide analogs each has a different fluorescent dyes thatdistinguishes each of the 4 different nucleotide analogs from eachother, wherein the 4 different nucleotides in the first set haveblocking moieties that can be selectively deblocked by a firsttreatment. A second set of 4 different nucleotide analogs each has adifferent fluorescent dyes that distinguishes each of the 4 differentnucleotide analogs from each other, wherein the 4 different nucleotidesin the second set have blocking moieties that can be selectivelydeblocked by a second treatment. In this case the first treatment isselective for the blocking moieties in the first set of nucleotides, andthe second treatment is selective for the blocking moieties in thesecond set of nucleotides. The two sets of dyes are unique such that the8 dyes produce 8 distinguishable signals, respectively.

In embodiments where all of the nucleotide analogs are distinguishablylabeled, such as the above-described 8 dye approach, a pair of templatesequences can be contacted with all of the nucleotide analogs and thendetection can be performed afterwards. Here the ability to distinguishall of the nucleotide analogs due to unique optical labels provides thebenefit of relatively simple fluidic manipulations, whereby all of thenucleotide analogs can be delivered to the template sequences such thatthey are simultaneously present. In a relatively straightforward andpreferred embodiment all 8 nucleotide analogs are deliveredsimultaneously; however, one or more subsets can be deliveredsequentially if desired. Detection can occur during or after nucleotideanalog delivery. This relatively simple fluidic process is accommodatedby a relatively complex detection device having the ability todistinguish all of the different signals. For example, a fluorescencedetection system able to distinguish 8 different fluorescent signals canbe used for the 8 dye approach Those skilled in the art will know or beable to determine an appropriate fluorescent detection apparatus toachieve this sort of signal differentiation. For example, excitation andemission properties of the fluorescent labels can be appropriatelymatched with a combination of excitation wavelengths produced andemission wavelengths detected by a fluorometer. Exemplary guides foroptics and labels useful for multiwavelength fluorescence detection areprovided in Lakowicz, Principles of Fluorescence Spectroscopy, 3^(rd)Ed. Springer (2006); Haugland, Handbook of Fluorescent Probes andResearch Products 9^(th) Ed., Molecular Probes, Inc, (2002); andShapiro, Practical Flow Cytometry, 4^(th) Ed., John Wiley & Sons (2003),each of which is incorporated herein by reference.

The principles exemplified above for a system in which all of thenucleotides are distinguishably labeled, can be readily extended to anarray format. An array having a sufficient number and variety ofdifferent template sequences will be expected to incorporate all of thelabeled nucleotides when treated with primer extension reaction systems.More specifically, in an array-based approach, having a wide variety ofnucleic acids across the array sites and having two different templatesper site, all possible 2-dye dye combinations will be expected to occuron the array following a primer extension cycle in which all 8nucleotides were delivered to the array. The sites can be spatiallydistinguished using optical devices known in the art, for example, thosedescribed in U.S. Pat. Nos. 8,241,573; 7,329,860 or 8,039,817; or USPat. App. Pub. Nos. 2009/0272914 A1 or 2012/0270305 A1, each of which isincorporated herein by reference. Such detection systems can be readilymodified to accommodate 8-color fluorescent detection as set forthabove. A detection system that is modified in this way will be capableof multiplex orthogonal detection such that two different templates aredistinguished (e.g. via sequencing) at multiple sites each having adifferent sequence composition.

In some embodiments, the number of different signals that aredistinguished in a particular method is less than the number ofdifferent nucleotide analog species used in that method. For example,multiple different nucleotide analog species can have the same labeland/or a subset of the nucleotide species can be unlabeled. An exampleof a configuration that uses the same label for multiple differentnucleotide species is the case of an orthogonal primer extension methodwhere a first set of 4 different nucleotide analogs share a first labelin common and are susceptible to a first selective deblocking treatment,whereas a second set of 4 different nucleotide analogs share a secondlabel in common and are susceptible to a second deblocking treatment. Inthis example the first label is optically distinguishable from thesecond label, the first treatment is selective for the first set ofnucleotide analogs and the second treatment is selective for the secondset of nucleotide analogs. In this configuration, the 4 differentnucleotides in the first set can be distinguished from each other bysequential cycles of delivery and detection (i.e. a separate cycle foreach of the different nucleotide analogs). So long as the first labeland second label in this example are distinguishable, members of the towsets of nucleotides can be delivered in pairs (1 each of a singlenucleotide species from the first set and a single nucleotide speciesfrom the second set), in 4 cycles of delivery and detection. Thus,members of a first set of nucleotide analogs used in a primer extensionreaction can include only one type of optical label that gets detectedand a second set of nucleotide analogs, that is orthogonal to the firstset can also include only one type of optical label that gets detected,wherein the label used in the first set is optically distinguishablefrom the label used in the second set.

Greyscaling allows use of multiple different nucleotide analog speciesthat have the same label. Here different nucleotide analog species canbe distinguished based on the intensity of label signal detected. Forexample, each species of nucleotide analog can be delivered as auniquely proportioned mixture of that species in labeled and unlabeledform. Variation in the ratio of labeled:unlabeled nucleotide analog foreach species will result in a uniquely greyscaled signal output for eachmixture. By way of more specific example, a first nucleotide analog canbe fully labeled (no mixing of labeled and unlabeled first nucleotideanalog), a second nucleotide analog can be 75% labeled (a mix of 75%labeled and 25% unlabeled second nucleotide analog), a third nucleotideanalog can be 50% labeled (a mix of 50% labeled and 50% unlabeled thirdnucleotide analog), and a fourth nucleotide analog can be 25% labeled (amix of 25% labeled and 75% unlabeled fourth nucleotide analog). These 4nucleotide analog species can be distinguished based on the resultingdifferences in signal intensity, whereby a population of appropriatelydeblocked primers (e.g. at an array site) will produce full signal dueto incorporation of the first nucleotide analog; 75% signal due toincorporation of the second nucleotide analog, 50% signal due toincorporation of the third nucleotide analog and 25% signal due toincorporation of the fourth nucleotide analog.

In particular embodiments, at least one of the nucleotide analog speciescan be entirely unlabeled. Thus, in a case where optical labels arepresent on the other nucleotide analogs in a set of nucleotide analogs,there can also be a ‘dark’ nucleotide analog. Extension of a primer toincorporate a dark, or otherwise unlabeled, nucleotide analog can bedetermined by inference based on the absence of a label that would beexpected if the other nucleotide analogs in the set were to have beenincorporated by the extension reaction. Thus, in some embodiments only asubset of the nucleotide analogs used in a primer extension reaction setforth herein need to have a label.

Use of entirely unlabeled nucleotide analog species can be combined withgreyscaling. For example, three of four different nucleotide analogspecies in a set (i.e. a set that can be deblocked by a commontreatment) can have distinguishable nonzero amounts of a particularlabel (e.g. ratios of labeled and non-labeled nucleotide analogs in amixture) and the fourth nucleotide analog species can lack that label.Alternatively or additionally, greyscaling can be combined with use ofseveral optically distinguishable labels. For example, some nucleotideanalog species can be represented in an extension reaction as a mixtureof nucleotides of the same type but having different labels. Such aconfiguration is exemplified in US Pat app. Pub. No. 2013/0079232 A1 or2015/0031560 A1, each of which is incorporated herein by reference.

Alternatively or additionally to the use of multiple different labels,greyscaling, and/or unlabeled species, an embodiment set forth hereincan use a nucleotide analog having a ligand, cleavable linker or othermoiety that provides for gain or loss of a label due to a definedtreatment. Reagent systems of this type are illustrated in US Pat App.Pub. Nos. 2013/0079232 A1 or 2015/0031560 A1 where some nucleotideanalog species have a ligand such that they can be distinguished fromother nucleotide analogs based on initial absence of a detectable signalfollowed by appearance of a signal after treatment with an appropriatelylabeled receptor. These references also illustrate use of a nucleotideanalog that can be distinguished based on an initial detectable signalthat is subsequently lost, or at least reduced, due to treatment with areagent that modifies the label (e.g. via chemical cleavage of a linkerbetween the label and nucleotide moieties). In this case the othernucleotide analog species in the set are not susceptible to themodification (e.g. lacking the cleavable linker) and are distinguishedbased on persistence of signal generation after the treatment.

As exemplified above, in some embodiments, a label can be attached to anucleotide analog via a cleavable linker. In particular embodiments,photocleavable linkers can be used in place of the chemically cleavablelinker exemplified above. In some embodiments, the linker is selectedfrom acid labile linkers (including dialkoxybenzyl linkers, Sieberlinkers, indole linkers, t-butyl Sieber linkers), electrophilicallycleavable linkers, nucleophilically cleavable linkers, photocleavablelinkers, linkers that are cleaved under reductive conditions oroxidative conditions, safety-catch linkers, and linkers that are cleavedby elimination mechanisms. In some such embodiments, the linker isselected from a disulfide linker (—S—S—), ester, nitrobenzene, imine,enzymatically or chemically cleavable peptide and polynucleotide, suchas DNA.

In some embodiments, members of a first set of nucleotide analogs (e.g.nucleotide analogs that are selectively deblocked by a first commontreatment) used in a primer extension reaction will include only onetype of optical label that gets detected and a second set of nucleotideanalogs (e.g. nucleotide analogs that are selectively deblocked by asecond common treatment), that is orthogonal to the first set will alsoinclude only one type of optical label that gets detected, wherein thelabel used in the first set is optically distinguishable from the labelused in the second set. In this embodiment, the one type of opticallabel can be attached to substantially all of the nucleotide analogs ofa first species in the first set, the one type of optical label can beattached to a subset of the nucleotide analogs of a second species inthe first set, substantially all of the nucleotide analogs of a thirdspecies in the first set can be attached to a ligand, and substantiallyall of the nucleotide analogs of a fourth species in the first set arenot attached to the one type of optical label or to the ligand.

In another embodiment, members of a first set of nucleotide analogs(e.g. nucleotide analogs that are selectively deblocked by a firstcommon treatment) used in a primer extension reaction will include onlytwo types of optical labels that get detected and a second set ofnucleotide analogs (e.g. nucleotide analogs that are selectivelydeblocked by a second common treatment), that is orthogonal to the firstset will also include only two types of optical label that get detected.In this embodiment, a first of the two types of optical labels can beattached to substantially all of the nucleotide analogs of a firstspecies in the first set, a second of the two types of optical labelscan be attached to substantially all of the nucleotide analogs of asecond species in the first set, the first of the two types of opticallabels and the second of the two types of optical labels can be attachedto nucleotide analogs of a third species in the first set, andsubstantially all of the nucleotide analogs of a fourth species in thefirst set are not attached to the one of the two types of optical labelsor the second of the two types of optical labels.

It will be understood from the above examples, that reducing the numberof different labels in an orthogonal detection system can provide theadvantage of reducing the complexity of the detection device needed todistinguish addition of different nucleotides to a template-boundprimer. However, in many embodiments this is achieved by increasing thecomplexity of the fluidic steps such that the number of fluidicmanipulations used during detection steps is increased compared to thefluidic steps used when each of the nucleotide species has a uniquelabel. A general advantage of the present methods is that one skilled inthe art can select an appropriate combination of labels, fluidic stepsand detection devices to suit a particular application or circumstance.

As exemplified by the embodiments set forth above, in some cases two ormore primers that are hybridized to two or more different nucleic acidtemplates, respectively, at a site can be simultaneously present duringa primer extension step. Alternatively, a first of two primers that arehybridized to two or more different templates at a site can be removedfrom the site prior to extending a second of the two or more primersthat are hybridized to the two or more different templates at the site.

Furthermore, two or more different primer extension products that resultfrom the above steps can be simultaneously present at a site during adetection step. Alternatively, a first of two or more different primerextension products can be removed from a site prior to detecting asecond of the two or more different primer extension products at thesite.

Further still, two or more primer extension products, each havingdifferent reversible blocking moieties, can be simultaneously presentduring a deblocking step. The deblocking step can be configured toselectively remove (or otherwise modify) one or only a subset of thedifferent reversible blocking moieties, such that at least one otherreversible blocking moiety is retained (or unmodified) following thetreatment. Alternatively, the different reversible blocking moietiesthat are simultaneously present can all be removed, for example, using acombination of deblocking treatments or a universal deblockingtreatment. As a further alternative, a first of two or more differentprimer extension products can be removed from a site prior to subjectinga second of the two or more primer extension products with a deblockingtreatment.

The present disclosure provides reaction mixtures (also referred toherein as reagent systems) that include various combinations ofcomponents. In several cases reaction components and severalcombinations of the components are described in the context of exemplarymethods, such as those set forth in the preceding paragraphs. It will beunderstood that the reaction mixtures and the components thereof neednot be limited to use in the methods exemplified herein. Other uses arecontemplated as well. Accordingly, the components can be assembled, in avariety of useful combinations, for example to create kits. The kits canbe useful for storage, transportation or commercial transaction of thecomponents set forth herein. The kits can optionally includeinstructions for carrying out one or more of the methods set forthherein.

In particular embodiments, this disclosure provides a method forsequencing nucleic acid templates that can include the steps of (a)providing an array of sites, wherein each site includes a first nucleicacid template and a second nucleic acid template, wherein the firstnucleic acid template has a sequence that is different from the sequenceof the second nucleic acid template, wherein a first primer is bound tothe first nucleic acid template, a reversible blocking moiety beingattached to the first primer, wherein a second primer is bound to thesecond nucleic acid template, a reversible blocking moiety beingattached to the second primer, and wherein the reversible blockingmoiety that is attached to the first primer is different from thereversible blocking moiety that is attached to the second primer; (b)selectively removing the reversible blocking moiety that is attached tothe first primer while retaining the reversible blocking moiety that isattached to the second primer; (c) extending the first primer byaddition of a first nucleotide analog that is attached to a reversibleblocking moiety; (d) selectively removing the reversible blocking moietythat is attached to the second primer while retaining the reversibleblocking moiety that is attached to the nucleotide analog that is addedto the first primer; (e) extending the second primer by addition of asecond nucleotide analog that is attached to a reversible blockingmoiety, wherein the reversible blocking moiety that is attached to thefirst nucleotide analog is different from the reversible blocking moietythat is attached to the second nucleotide analog; and (f) detecting thenucleotide analog that is added to the first primer and the nucleotideanalog that is added to the second primer, at each of the sites, therebydetermining the different sequences of the first template and the secondtemplate at each of the sites.

In some embodiments, the above method can further include steps of (g)selectively removing the reversible blocking moiety that is attached tothe first nucleotide analog that is added to the first primer whileretaining the reversible blocking moiety that is attached to the secondnucleotide analog that is added to the second primer; (h) extending thefirst primer, after (g), by addition of a third nucleotide analog thatis attached to a reversible blocking moiety; (i) selectively removingthe reversible blocking moiety that is attached to the second nucleotideanalog that is added to the second primer while retaining the reversibleblocking moiety that is attached to the first nucleotide analog that isadded to the first primer; (j) extending the second primer, after (i),by addition of a fourth nucleotide analog that is attached to areversible blocking moiety, wherein the reversible blocking moiety thatis attached to the third nucleotide analog is different from thereversible blocking moiety that is attached to the fourth nucleotideanalog; and (k) detecting the nucleotide analog that is added to thefirst primer in (h) and the nucleotide analog that is added to thesecond prime in (j), at each of the sites, thereby determining thedifferent sequences of the first template and the second template ateach of the sites. Optionally, steps (g) through (k) can be repeated.

It will be understood that the steps set forth in the above embodimentand other embodiments of the present disclosure, need not follow theexemplified order. Taking as an example the embodiment in the precedingparagraphs, step (f), which recites a detection activity, need not occurafter step (e). Rather, a first primer that is extended in step (c) canbe detected prior to extending a second primer in step (d). Generally, adetection step can occur before or after one or more differentreversible blocking moieties are removed.

The order of other steps can be changed to suit particular applicationsof the methods. An example of two methods that can employ similar steps,but in different orders is demonstrated by comparison of the cyclediagrams in FIG. 1 and FIG. 2. In the cycle diagram of FIG. 1 labels onprimer extension products are detected prior to both deblocking andcleavage of the labels. Deblocking and label cleavage for nucleotideswithin each set are shown as happening simultaneously, which can beconvenient, for example, when both are susceptible to the same treatmentor due to compatible treatments. However, it is possible for deblockingand label cleavage to occur separately. As exemplified by the cyclediagram of FIG. 2, label cleavage and deblocking occur separately. Inthis example, the different labels that are present across the twodifferent reactions (i.e. R1 and R2) are cleaved simultaneously. This isconvenient, for example, when both types of labels can be cleaved usingthe same treatment or using compatible treatments. Comparison of thecycle diagrams in FIG. 1 and FIG. 2 illustrate other differences aswell. For example, in FIG. 1, deblocking of the extension products ofboth reactions (R1 and R2) occurs after the detection step of theprevious cycle, whereas in FIG. 2, the R2 extension product is deblockedprior to the detection step of the previous cycle and the R1 extensionproduct is deblocked after the detection step of the previous cycle.There are a variety of different arrangements and orders of steps thatcan be used in a method set forth herein. Those skilled in the art willbe able to readily determine a desirable arrangement and order based onthe teaching set forth herein and known reactive characteristics of thereagents employed.

Furthermore, as exemplified previously herein the steps of the methodsset forth herein can be carried out sequentially or simultaneously. Forexample, the selective removal of different reversible blocking moieties(e.g. steps (b) and (d) in the preceding paragraphs) can be carried outsimultaneously or sequentially. Similarly, the extension of differentprimers (e.g. steps (c) and (e) in the preceding paragraphs), that havebeen deblocked using respective different deblocking treatments, canoccur simultaneously or sequentially.

Universal priming sites are particularly useful for multiplexapplications of the methods set forth herein. A universal priming siteprovides a region of sequence that is common to two or more nucleic acidmolecules where the molecules also have different sequences in theirtemplate regions. A universal priming sequence present in differentmembers of a collection of molecules can allow the replication,amplification or detection of multiple different sequences using asingle universal primer species that is complementary to the universalsequence. Examples of methods of attaching universal sequences to acollection of target nucleic acids can be found in US Pat. App. Pub. No.2007/0128624 A1, which is incorporated herein by reference.

In embodiments of the present disclosure, a first primer can have afirst universal primer sequence that is complementary to a firstuniversal priming site for a first template at a site. The sameuniversal priming site can be present for a variety of different firsttemplates at different sites of an array. Thus a single species of firstuniversal primer can be used to amplify or extend the different firsttemplates at the sites. Continuing with this example, a second primercan have a second universal primer sequence that is complementary to asecond universal priming site for a second template at the site wherethe first template is also located. The same second universal primingsite can be present for a variety of different second templates at thedifferent sites of the array. Thus, a single species of second universalprimer can be used to amplify or extend the different second templatesat the sites.

An orthogonal sequencing method set forth herein can be utilized in apaired-end sequencing approach. Generally, paired end sequencinginvolves determining the sequences at two ends of a template sequenceregion, wherein the length of the template sequence region is known.Methods for fragmenting a target nucleic acid sample (e.g. genomic DNAsample), attaching primers to accommodate paired end reads and readingsequence from the ends of the fragments are known and can be carried outas described, for example, in U.S. Pat. Nos. 7,754,429; 8,017,335 or8,192,930, each of which is incorporated herein by reference.

In the case of a sequencing-by-synthesis embodiment, nucleic acidfragments can be constructed to have two template sequences and pairedreads can be obtained from each of the two templates to obtain 4 readsfrom a single fragment. An exemplary construct for obtaining 4 readsfrom a single fragment and methods for making the construct are setforth in US Pat. App. Pub. No. 2015/0031560 A1, which is incorporatedherein by reference.

The present disclosure further provides a nucleic acid array thatincludes a plurality of sites on a solid support, wherein each siteincludes a first nucleic acid template and a second nucleic acidtemplate, wherein the first nucleic acid template has a sequence that isdifferent from the sequence of the second nucleic acid template, whereina first primer is bound to the first nucleic acid template, a firstreversible blocking moiety being attached to the first primer, wherein asecond primer is bound to the second nucleic acid template, a secondreversible blocking moiety being attached to the second primer, andwherein the first reversible blocking moiety is different from thesecond reversible blocking moiety. The nucleic acid array can furtherinclude one or more of the components described in the context ofmethods of the present disclosure. Products that inherently result fromthe methods set forth herein are also intended to be considered ascomponents of a nucleic acid in some embodiments.

In particular embodiments, a nucleic acid array will include a firstpolymerase that is bound to the first primer and the first nucleic acidtemplate at a site. Additionally, a second polymerase can be bound tothe second primer and the second nucleic acid template at the site. Insome cases, the first polymerase and second polymerase are the samespecies of polymerase. However, it can also be useful in someembodiments, for the first and second polymerases to be differentspecies (e.g. a DNA polymerase and an RNA polymerase).

A nucleic acid array of the present disclosure can be present in adetection apparatus such as a nucleic acid sequencing apparatus.Exemplary detection apparatus are described herein and in referencesthat are incorporated herein by reference. Generally, a detectionapparatus can include a nucleic acid array and a detector that ispositioned to detect one or more sites in the array. Typically, thedetector will have a spatial resolution that is too low to resolvepoints at distance equivalent to the spacing between the first primerand the second primer at each of the sites. However, the use oforthogonal primer deblocking and extension allows the two primers to beresolved. The detector can be configured to observe any of a variety ofsignals as exemplified herein. For example, in some embodiments thedetector is an optical detector. The sites of the array can have opticallabels that are detectable by the optical detector. Different primers ateach site can be extended to incorporate different optical labels andthe optical detector can be configured to optically distinguish thedifferent labels (e.g. due to differences in wavelength of lightabsorption, wavelength of luminescence excitation or wavelength ofluminescence emission). In some configurations, a pixel of the detectoris configured to simultaneously acquire signals from the first primerand the second primer.

Throughout this application various publications, patents or patentapplications have been referenced. The disclosure of these publicationsin their entireties are hereby incorporated by reference in thisapplication.

The term comprising is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the following claims.

What is claimed is:
 1. A nucleic acid array, comprising a plurality ofsites on a solid support, wherein each site comprises a first nucleicacid template and a second nucleic acid template, wherein the firstnucleic acid template has a sequence that is different from the sequenceof the second nucleic acid template, wherein a first primer is bound tothe first nucleic acid template, a first reversible blocking moietybeing attached to the first primer, wherein a second primer is bound tothe second nucleic acid template, a second reversible blocking moietybeing attached to the second primer, and wherein the first reversibleblocking moiety is different from the second reversible blocking moiety.2. The nucleic acid array of claim 1, wherein each site occupies an areaon the solid support that is 100 μm² or less.
 3. The nucleic acid arrayof claim 1, wherein the plurality of sites has a pitch of 10 μm or less.4. The nucleic acid array of claim 1, wherein the plurality of sitescomprises at least 1×10⁶ sites.
 5. The nucleic acid array of claim 4,wherein each of the sites comprises a nucleic acid sequence that isunique compared to the nucleic acid sequences at the other sites in theplurality.
 6. The nucleic acid array of claim ′, wherein the firstreversible blocking moiety is attached to the 3′ nucleotide of the firstprimer.
 7. The nucleic acid array of claim 6, wherein the 3′ nucleotideof the first primer is attached to a first optical label.
 8. The nucleicacid array of claim 1, wherein the second reversible blocking moiety isattached to the 3′ nucleotide of the second primer.
 9. The nucleic acidarray of claim 8, wherein the 3′ nucleotide of the second primer isattached to a second optical label, wherein the second optical label isoptically distinguishable from the first optical label.
 10. The nucleicacid array of claim 1, wherein the first and second optical labelscomprise fluorophores.
 11. The nucleic acid array of claim 1, whereinthe first nucleic acid template and the second nucleic acid templatecomprise DNA.
 12. The nucleic acid array of claim 1, wherein a singlenucleic acid molecule contains the first nucleic acid template and thesecond nucleic acid template.
 13. The nucleic acid array of claim 1,wherein the first nucleic acid template and the second nucleic acidtemplate are on different nucleic acid molecules.
 14. The nucleic acidarray of claim 1, wherein the sites comprise multiple amplicons of thefirst nucleic acid template and multiple amplicons of the second nucleicacid template.
 15. The nucleic acid array of claim 14, wherein themultiple amplicons comprise a nucleic acid cluster.
 16. The nucleic acidarray of claim 1, wherein the first primer comprises a first universalprimer sequence and the first nucleic acid template at each site in theplurality of sites comprises a first universal primer binding sequencethat is complementary to the first universal primer sequence.
 17. Thenucleic acid array of claim 16, wherein the second primer comprises asecond universal primer sequence and the second nucleic acid template ateach site in the plurality of sites comprises a second universal primerbinding sequence that is complementary to the second universal primersequence, wherein the first universal primer binding sequence isdifferent from the second universal primer binding sequence.
 18. Thenucleic acid array of claim 1, wherein a first polymerase is bound tothe first primer and the first nucleic acid template.
 19. The nucleicacid array of claim 18, wherein a second polymerase is bound to thesecond primer and the second nucleic acid template, and wherein thefirst polymerase and second polymerase are the same species ofpolymerase.
 20. The nucleic acid array of claim 1, wherein the firstreversible blocking moiety comprises azidomethyl and wherein the secondreversible blocking moiety comprises tert-butoxy-ethoxy.
 21. A detectionapparatus, comprising the nucleic acid array of claim 1, and a detectorpositioned to detect the plurality of sites.
 22. The detection apparatusof claim 21, wherein the detector has a spatial resolution that is toolow to resolve points at distance equivalent to the spacing between thefirst primer and the second primer at each of the sites.
 23. Thedetection apparatus of claim 21, wherein the detector is an opticaldetector.
 24. The detection apparatus of claim 21, wherein a pixel ofthe detector is configured to simultaneously acquire signals from thefirst primer and the second primer.